The Preliminary Study For Rapid Differential Diagnosis Of Drug-resistant TB By Use Of PMA-qPCR

Objective:To establish a rapid drug susceptibility testing technology,shorten the time for obtaining results and provide timely and proper treatment for patients,PMA-qPCR technology is applied to the drug resistant tuberculosis diagnosis in the hope of making a rapid detection for drug resistance of Mycobacterium tuberculosis.Methods:First,we design premers according to the conserved sequence of Mycobacterium tuberculosis16sRNA gene published in GenBank and the amount of initial template is considered as the standard of PMA inhibiting amplification of bacteria. We dilute PMA to0.5mg/ml with DMSO and optimize and select the following experimental conditions after interaction of PMA and Mycobacterium tuberculosis.(1)After interaction between3ug/ml PMA and single bacterial suspension,we set different exposure time and select the optimal exposure time of PMA as the exposure time in subsequent tests.(2)We make dead mycobacterium tuberculosis cells with heat inactivated, ultrasonic lysis and UV irradiation and select the best method from the three lethal method after exposuring to halogen and qPCR analysis of CT values.(3)After interaction between different concentrations of PMA and viable bacteria,we select minimum concentration of PMA not inhibiting amplification of viable bacteria.(4)We select the minimum concentration of PMA inhibiting amplification of dead bacteria.(5)Determination of the minimum percentage of dead bacteria after interation of3ug/ml PMA and different proportion of live/dead bacteria mixture.(6)The mycobacterium tuberculosis are cultured3days in treatment with anti-tuberculosis drug and PMA,and then determine the drug resistance of Mycobacterium tuberculosis accordding to the differences of CT values (△CT or2△CT) between the control and drug groups.Results:(1)The16sRNA gene of Mycobacterium tuberculosis has species specificity and the primer dimers are not obvious which does not affect the analysis of qPCR and could be used to PMA-qPCR test.(2) The best exposure time of PMA is15min.(3)The best lethal method of mycobacterium tuberculosis is Heat-inactivated.(4) The minimum concentration of PMA not inhibiting amplification of living mycobacterium tuberculosis bacteria is20ug/ml.(5)The optimal concentration of PMA completely inhibiting amplification of dead bacteria is3ug/ml.(6) PMA could effectively distinguish and detect living bacteria from the mixture of live and dead bacteria when the dead bacteria account for50%-80%.(7)PMA-qPCR can effectively distinguish the drug sensitivity of MTB.Conclusion:We established the method for the detection of drug resistance of MTB and shorten the detection time and the technology was expected to become a standard detection for the drug resistance tuberculosis.